Analysis of protooncogene activation in rodent and human tumors has been the major focus of this project. In a previous study, we detected activated protooncogenes in 12 of 14 lung tumors from smokers. This study, two non-ras genes are being cloned. Approximately 26 kbp has been cloned from the first non-ras gene, appears to correspond to a novel protooncogene and it has been localized to chromosome 10p by in situ hybridization. The cloned sequences have been used to obtain a cDNA clone that gives tumors in the nude mouse tumorigenicity assay. A single human DNA fragment of 16 kbp has been cloned from the second non- ras gene and used to localize this gene to chromosome 20 by in situ hybridization. An additional set of human pulmonary squamous cell carcinomas have been analyzed for activated protooncogenes using the nude mouse tumorigenicity assay. Second cycle transfections indicated that 5 of 16 of these squamous cell carcinomas contained detectable transforming genes that were not H-, K-, or N-ras or c-raf genes as determined by Southern blot analysis. Rat lung tumors from animals treated with diethylnitrosamine (DEN) and promoted with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) scored positive in the tumorigenicity assay for oncogenes. The analysis of highly conserved DNA fragments that contained rat repetitive sequences suggested that the same gene was activated in each of the adenocarcinomas. To further explore the genetics of the K-ras gene in mouse lung tumor susceptibility, the parental origin of K-ras oncogenes detected in lung tumors from F1 hybrids was determined. K-ras oncogenes were derived from the susceptible A/J parent in 38/40 tumors from C3A hybrid mice and 30/30 tumors from AC3 hybrid mice. The observation that the oncogene in hybrids originates from the susceptible parent suggests that the K-ras gene is directly linked to mouse lung tumor susceptibility. Examination of the promoter regions and the regions surrounding the 37 bp repeat in the C3H strain may enable us to determine the mechanism of allele specific activation of the A/J K-ras gene.